Enhanced Protein Mass Spectrometry Analysis with …...Title Enhanced Protein Mass Spectrometry...

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Missed R Missed R

Missed K (80-90% of missed

cleavages)

Missed K

Marjeta Urh1, Sergei Saveliev1, Ethan Strauss1, Mike Rosenblatt1, Richard Jones2, Michael Ford2

and Dave Allen2

1Promega Corporation, Madison, WI; 2MS BioWorks, Ann Arbor, MI

1. Introduction 2. Text goes here

It can be 2 lines

3. Text goes here

It can be 2 lines

6. Text goes here

It can be 2 lines 2. Proteolytic enhancement with Trypsin/Lys-C

3. Trypsin/Lys-C advantage for mass spec analysis 4. An additional advantage: digestion of

proteolytically resistant proteins

5. Conclusions

Incomplete proteolysis in trypsin digests is largely attributed

to trypsin proteolytic inefficiency at lysine sites. Missed K and missed R ratio in tryptic digests equals to 5:1-6:1

Supplementing trypsin with Lys-C leads to elimination of the

majority of missed lysine sites and overall increase in

proteolytic efficiency.

Trypsin/Lys-C mix tolerates protease inhibiting impurities

and digests tightly folded, proteolytically resistant proteins* *Digestion of proteolytically resistant proteins requires a specialized, two-

step procedure

Enhanced digestion with Trypsin/Lys-C provides critical

benefits for protein mass spec analysis: Increase in peptide and protein identifications

Higher analytical reproducibility

More accurate protein quantitation through more complete digestion

Enhanced protein mass spectrometry analysis with Trypsin/Lys-C mix

Efficient proteolysis is required for protein mass spectrometry

analysis. Here we describe a Trypsin/Lys-C mix, which dramatically

improves proteolysis:

the majority of missed cleavages, commonly occurring in trypsin

digests, are largely eliminated

protease-inhibiting impurities are tolerated

proteolytically resistant proteins are efficiently digested

We show here how enhanced proteolysis with Trypsin/Lys-C

improves protein mass spectrometry analysis. These benefits include

an increase in identified peptides and proteins, higher signal intensity

of individual peptides and higher analytical reproducibility.

Digested sample Missed K

Missed R

Missed K: Missed R

ratio

Yeast extract 18.6% 3.6% 5.5:1

Mouse extract 6.6% 1.1% 6:1

In a typical trypsin digest,

10-30% cleavage sites are

‘missed’ (undigested).

Majority of missed

cleavages are lysines.

Add trypsin/Lys-C Add Urea to 6-8M Dilute the reaction and continue

digestion

Digestion of proteolytically

resistant protein (myoglobin)

with Trypsin/Lys-C

Digestion with Trypsin

(myoglobin remains intact)

Digestion with Trypsin/Lys-C

(myoglobin is efficiently digested)

Intact

protein

Peptides

RP HPLC spectra

Protein

denatures. It is

now amenable to

proteolysis

Lys-C digests a protein

onto relatively large

fragments. Trypsin is

inactivated.

Trypsin re-activates

and completes

digestion

Protein is

resistant to

trypsin due to

tight

conformation

Alternative protocol; a two step digestion.

24% and 10% increase of identified peptides and proteins, respectively.

40% and 17% increase of identified

peptides and proteins, respectively.

NNNNNK NNNNNR NNNNNN

Trypsin/Lys-C cleavage specificity

705 887

Trypsin digest

Trypsin/Lys-C digest

FFPE skin tissue

Identified peptides

1490 2092

Arabidopsis extract

Protein

Protein mass spec analysis

Peptides

Trypsin/Lys-C eliminates the

majority of missed lysine

cleavages and increases

overall proteolytic efficiency.

Digested sample

Missed K

Missed R

Missed K: Missed R

ratio

Yeast extract 2.6% 4% 0.65:1

Mouse extract 2.9% 1.5% 1.9:1

NNNNNK NNNNNR NNNNNN

Trypsin cleavage specificity

0

10

20

IgG (Rituximab) digestion reproducibility

CV peptide peak area (n=10)

SLSLSPGK ALPAPIEK QVQLQQPGAELVK

Trypsin digests (n=10)

Trypsin/Lys-C digests (n=10)

Low CV in Trypsin/Lys-C digest

Increase in peptide and protein identifications

Higher peptide recovery

Trypsin digest

Trypsin digest

Trypsin/Lys-C digest Trypsin/Lys-C

digest 1.7-4.6 increase in peptide peak area. Improved protein quantitation!

Quantitation of Serum Amyloid P component (SAMP) protein in plasma

Higher digestion reproducibility

Improved digestion reproducibility

Tolerance to protease inhibiting agents

0

30

60

1252 1204

1495

1364

Digestion in the presence of protease inhibitors and denaturants Yeast protein extract was used as a model substrate

30%

0%

60%

Missed cleavages Identified proteins

Trypsin digest

Trypsin/Lys-C digest

Efficient digestion in the presence of inhibiting agents accompanied by an increase in protein identifications

10%

0%

20%

Trypsin/Lys-C lowers the level of missed cleavages to norm

The number of identified proteins increases by up to 20%

GuCl, 0.5M Protease inhibitor cocktail, 1x

Courtesy by Dr. Szapacs, GSK

Courtesy by Dr. Adams, Stanford University Courtesy by Dr. Minkoff, University of Wisconsin-Madison

Similar ratios of missed lysine and arginine cleavages sites were observed in Trypsin and Trypsin/Lys-C digests of extracts prepared from other sources including human and E.coli.

Regular one-step overnight digestion with Trypsin

Regular one-step overnight digestion with Trypsin/Lys-C mix

GuCl, 0.5M Protease inhibitor cocktail, 1x

Trypsin digest

Trypsin/Lys-C digest

Identified peptides

Peptide peak area Peptide peak area

Trypsin/Lys-C

CV

peptide p

eak a

rea

(n=

10)